Single turnover kinetics of phage T4 DNA-(N6-adenine)methyltransferase

Interaction of T4 DNA-(N6-adenine)-methyltransferase [EC 2.1.1] was studied with a variety of synthetic oligonucleotide substrates containing the native recognition site GATC or its modified variants. The data obtained in the decisecond and second intervals of the reaction course allowed for the first time the substrate methylation rates to be compared with the parameters of the steady-state reaction. It was established that the substrate reaction proceeds in two stages. Because it is shown that in steady-state conditions T4 MTase forms a dimeric structure, the following sequence of events is assumed. Upon collision of a T4 MTase monomer with an oligonucleotide duplex, an asymmetrical complex forms in which the enzyme randomly oriented relative to one of the strands of the specific recognition site catalyzes a fast transfer of the methyl group from S-adenosylmethionine to the adenosine residue (k1 = 0.21 s-1). Simultaneously, a second T4 MTase subunit is added to the complex, providing for the continuation of the reaction. In the course of a second stage, which is by an order of magnitude slower (k2 = 0.023 s-1 for duplex with the native site), the dimeric T4 MTase switches over to the second strand and the methylation of the second residue, target. The rate of the methyl group transfer from donor, S-adenosylmethionine, to DNA is much higher than the overall rate of the T4 MTase-catalyzed steady-state reaction, although this difference is considerably less than that shown for EcoRI Mtase. Substitutions of bases and deletions in the recognition site affect the substrate parameters in different fashions. When the GAT sequence is disrupted, the proportion of the initial productive enzyme-substrate complexes is usually sharply reduced. The flipping of the adenosine residue, a target for the modification in the recognition site, revealed by fluorescence titration, upon interaction with the enzyme supports the existing notions about the involvement of such a DNA deformation in reactions catalyzed by various DNA-MTases.     

Formation of a covalent complex between methylguanine methyltransferase and DNA via disulfide bond formation between the active site cysteine and a thiol-containing analog of guanine.

DNA repair methyltransferases (MTases) remove methyl or other alkyl groups from the O6 position of guanine or the O4 position of thymine by transfering the group to an active site cysteine. In order to trap an MTase-DNA complex via a disulfide bond, 2'-deoxy-6-(cystamine)-2-aminopurine (d6Cys2AP) was synthesized and incorporated into oligonucleotides. d6Cys2AP has a disulfide bond within an alkyl chain linked to the 6 position of 2,6-diaminopurine, which disulfide can be reduced to form a free thiol. Addition of human MTase to reduced oligonucleotide resulted in a protein-DNA complex that was insensitive to denaturation by SDS and high salt, but which readily dissociated in the presence of dithiothreitol. Formation of this complex was prevented by methylation of the active site cysteine. Evidence that the active site cysteine is directly involved in disulfide bond formation was obtained by N-terminal sequencing of peptides that remained associated with DNA after proteolysis of the complex.     

Self-methylation of BspRI DNA-methyltransferase.

The DNA (cytosine-5)-methyltransferase (m5C-MTase) M.BspRI is able to accept the methyl group from the methyl donor S-adenosyl-L-methionine (AdoMet) in the absence of DNA. Transfer of the methyl group to the enzyme is a slow reaction relative to DNA methylation. Self-methylation is dependent on the native conformation of the enzyme and is inhibited by S-adenosyl-L-homocysteine, DNA and sulfhydryl reagents. Amino acid sequencing of proteolytic peptides obtained from M.BspRI, which had been methylated with [methyl-3H]AdoMet, and thin layer chromatography of the modified amino acid identified two cysteines, Cys156 and Cys181 that bind the methyl group in form of S-methylcysteine. One of the acceptor residues, Cys156 is the highly conserved cysteine which plays the role of the catalytic nucleophile of m5C-MTases.     

Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes

Gene silencing by targeted DNA methylation has potential applications in basic research and therapy. To establish targeted methylation in human cell lines, the catalytic domains (CDs) of mouse Dnmt3a and Dnmt3b DNA methyltransferases (MTases) were fused to different DNA binding domains (DBD) of GAL4 and an engineered Cys2His2 zinc finger domain. We demonstrated that (i) Dense DNA methylation can be targeted to specific regions in gene promoters using chimeric DNA MTases. (ii) Site-specific methylation leads to repression of genes controlled by various cellular or viral promoters. (iii) Mutations affecting any of the DBD, MTase or target DNA sequences reduce targeted methylation and gene silencing. (iv) Targeted DNA methylation is effective in repressing Herpes Simplex Virus type 1 (HSV-1) infection in cell culture with the viral titer reduced by at least 18-fold in the presence of an MTase fused to an engineered zinc finger DBD, which binds a single site in the promoter of HSV-1 gene IE175k. In short, we show here that it is possible to direct DNA MTase activity to predetermined sites in DNA, achieve targeted gene silencing in mammalian cell lines and interfere with HSV-1 propagation.     

Kinetic and catalytic properties of dimeric KpnI DNA methyltransferase

KpnI DNA-(N(6)-adenine)-methyltransferase (KpnI MTase) is a member of a restriction-modification (R-M) system in Klebsiella pneumoniae and recognizes the sequence 5'-GGTACC-3'. It modifies the recognition sequence by transferring the methyl group from S-adenosyl-l-methionine (AdoMet) to the N(6) position of adenine residue. KpnI MTase occurs as a dimer in solution as shown by gel filtration and chemical cross-linking analysis. The nonlinear dependence of methylation activity on enzyme concentration indicates that the functionally active form of the enzyme is also a dimer. Product inhibition studies with KpnI MTase showed that S-adenosyl-l-homocysteine is a competitive inhibitor with respect to AdoMet and noncompetitive inhibitor with respect to DNA. The methylated DNA showed noncompetitive inhibition with respect to both DNA and AdoMet. A reduction in the rate of methylation was observed at high concentrations of duplex DNA. The kinetic analysis where AdoMet binds first followed by DNA, supports an ordered bi bi mechanism. After methyl transfer, methylated DNA dissociates followed by S-adenosyl-l-homocysteine. Isotope-partitioning analysis showed that KpnI MTase-AdoMet complex is catalytically active.     

Characterization of the major DNA repair methyltransferase activity in unadapted Escherichia coli and identification of a similar activity in Salmonella typhimurium.

Escherichia coli has two DNA repair methyltransferases (MTases): the 39-kilodalton (kDa) Ada protein, which can undergo proteolysis to an active 19-kDa fragment, and the 19-kDa DNA MTase II. We characterized DNA MTase II in cell extracts of an ada deletion mutant and compared it with the purified 19-kDa Ada fragment. Like Ada, DNA MTase II repaired O6-methylguanine (O6MeG) lesions via transfer of the methyl group from DNA to a cysteine residue in the MTase. Substrate competition experiments indicated that DNA MTase II repaired O4-methylthymine lesions by transfer of the methyl group to the same active site within the DNA MTase II molecule. The repair kinetics of DNA MTase II were similar to those of Ada; both repaired O6MeG in double-stranded DNA much more efficiently than O6MeG in single-stranded DNA. Chronic pretreatment of ada deletion mutants with sublethal (adapting) levels of two alkylating agents resulted in the depletion of DNA MTase II. Thus, unlike Ada, DNA MTase II did not appear to be induced in response to chronic DNA alkylation at least in this ada deletion strain. DNA MTase II was much more heat labile than Ada. Heat lability studies indicated that more than 95% of the MTase in unadapted E. coli was DNA MTase II. We discuss the possible implications of these results for the mechanism of induction of the adaptive response. A similarly active 19-kDa O6MeG-O4-methylthymine DNA MTase was identified in Salmonella typhimurium.     

Comparative studies of the phage T2 and T4 DNA (N6-adenine)methyltransferases: amino acid changes that affect catalytic activity.

The bacteriophage T2 and T4 dam genes code for a DNA (N6-adenine)methyltransferase (MTase). Nonglucosylated, hydroxymethylcytosine-containing T2gt- virion DNA has a higher level of methylation than T4gt- virion DNA does. To investigate the basis for this difference, we compared the intracellular enzyme levels following phage infection as well as the in vitro intrinsic methylation capabilities of purified T2 and T4 Dam MTases. Results from Western blotting (immunoblotting) showed that the same amounts of MTase protein were produced after infection with T2 and T4. Kinetic analyses with purified homogeneous enzymes showed that the two MTases had similar Km values for the methyl donor, S-adenosyl-L-methionine, and for substrate DNA. In contrast, they had different k(cat) values (twofold higher for T2 Dam MTase). We suggest that this difference can account for the ability of T2 Dam to methylate viral DNA in vivo to a higher level than does T4 Dam. Since the T2 and T4 MTases differ at only three amino acid residues (at positions 20 [T4, Ser; T2, Pro], 26 [T4, Asn; T2, Asp], and 188 [T4, Asp; T2, Glu]), we have produced hybrid proteins to determine which residue(s) is responsible for increased catalytic activity. The results of these analyses showed that the residues at positions 20 and 26 are responsible for the different k(cat) values of the two MTases for both canonical and noncanonical sites. Moreover, a single substitution of either residue 20 or 26 was sufficient to increase the k(cat) of T4 Dam.     

Zinc binding by the methylation signaling domain of the Escherichia coli Ada protein.

The Escherichia coli Ada protein repairs O6-methylguanine residues and methyl phosphotriesters in DNA by direct transfer of the methyl group to a cysteine residue located in its C- or N-terminal domain, respectively. Methyl transfer to the N-terminal domain causes it to acquire a sequence-specific DNA binding activity, which directs binding to the regulatory region of several methylation-resistance genes. In this paper we show that the N-terminal domain of Ada contains a high-affinity binding site for a single zinc atom, whereas the C-terminal domain is free of zinc. The metal-binding domain is apparently located within the first 92 amino acids of Ada, which contains four conserved cysteine residues. We propose that these four cysteines serve as the zinc ligand residues, coordinating the metal in a tetrahedral arrangement. One of the putative ligand residues, namely, Cys69, also serves as the acceptor site for a phosphotriester-derived methyl group. This raises the possibility that methylation-dependent ligand reorganization about the metal plays a role in the conformational switching mechanism that converts Ada from a non-sequence-specific to a sequence-specific DNA-binding protein.     

Single Turnover Kinetics of Methylation by T4 DNA-(N6-Adenine)-Methyltransferase

Interaction of T4 DNA-(N6-adenine)-methyltransferase was studied with a variety of synthetic oligonucleotide substrates containing the native recognition site GATC or its modified variants. The data obtained in the decisecond and second intervals of the reaction course allowed for the first time the substrate methylation rates to be compared with the parameters of the steady-state reaction. It was established that the substrate reaction proceeds in two stages. Because it is shown that in steady-state conditions T4 MTase forms a dimeric structure, the following sequence of events is assumed. Upon collision of a T4 MTase monomer with an oligonucleotide duplex, an asymmetrical complex forms in which the enzyme randomly oriented relative to one of the strands of the specific recognition site catalyzes a fast transfer of the methyl group from S-adenosylmethionine to the adenosine residue (k ₁ = 0.21 s^–1). Simultaneously, a second T4 MTase subunit is added to the complex, providing for the continuation of the reaction. In the course of a second stage, which is by an order of magnitude slower (k ₂ = 0.023 s^–1 for duplex with the native site), the dimeric T4 MTase switches over to the second strand and the methylation of the second residue, target. The rate of the methyl group transfer from donor, S-adenosylmethionine, to DNA is much higher than the overall rate of the T4 MTase-catalyzed steady-state reaction, although this difference is considerably less than that shown for EcoRI MTase. Base substitutions and deletions in the recognition site affect the substrate parameters in different fashions. When the GAT sequence is disrupted, the proportion of the initial productive enzyme–substrate complexes is usually sharply reduced. The flipping of the adenosine residue to be modified in the recognition site upon interaction with the enzyme, revealed by fluorescence titration, supports the existing notions about the involvement of such a DNA deformation in reactions catalyzed by various DNA-MTases.     

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5’-ATCGAT-3’ sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.     

Geometry of interaction of metal ions with sulfur-containing ligands in protein structures

An analysis of the geometry of binding of metal ions by cysteine and methionine residues in protein structures has been made by using the Protein Data Bank. Metal ions have a distinct model of binding to each of these residues, and this is independent of the nature of the metal center or the type of protein. Metal ions tend to approach the sulfur of Met roughly 38 degrees from the perpendicular to the plane through atoms C gamma-S delta-C epsilon. For Cys, the approach direction is such that the M...S gamma-C beta-C alpha torsional angle is about +/- 90 or 180 degrees. The side-chain conformation of the cysteine residue is affected by the presence of the metal ion; there is a shift from the g+ conformation toward g- and mainly t conformations. When two Cys residues at positions i-3 and i bind to the same metal center, there appears to be some restriction on the geometry of metal binding by the residue i; for such a residue chi 1 and M...S gamma-C beta-C alpha angles are likely to be around 60 degrees and 270 degrees, respectively. Met and Cys residues coordinating to a metal ion are usually from coil or turn regions of the protein structure.     

Purification and characterization of rice DNA methyltransferase

Epigenetic modification is essential for normal development and plays important roles in gene regulation in higher plants. Multiple factors interact to regulate the establishment and maintenance of DNA methylation in plant genome. We had previously cloned and characterized DNA methyltransferase (DNA MTase) gene homologues (OsMET1) from rice. In this present study, determination of DNA MTase activity in different cellular compartments showed that DNA MTase was enriched in nuclei and the activity was remarkably increased during imbibing dry seeds. We had optimized the purification technique for DNA MTase enzyme from shoots of 10-day-old rice seedlings using the three successive chromatographic columns. The Econo-Pac Q, the Hitrap-Heparin and the Superdex-200 columns yielded a protein fraction of a specific activity of 29, 298 and 800 purification folds, compared to the original nuclear extract, respectively. The purified protein preferred hemi-methylated DNA substrate, suggesting the maintenance activity of methylation. The native rice DNA MTase was approximately 160–170 kDa and exhibited a broad pH optimum in the range of 7.6 and 8.0. The enzyme kinetics and inhibitory effects by methyl donor analogs, base analogs, cations, and cationic amines on rice DNA MTase were examined. Global cytosine methylation status of rice genome during development and in various tissue culture systems were monitored and the results suggested that the cytosine methylation level is not directly correlated with the DNA MTase activity. The purification and characterization of rice DNA MTase enzyme are expected to enhance our understanding of this enzyme function and their possible contributions in Gramineae plant development.     

DNA-(N4-cytosine)-methyltransferase from Bacillus amyloliquefaciens: kinetic and substrate binding properties

Interaction of DNA-(N4-cytosine)-methyltransferase from the Bacillus amyloliquefaciens (BamHI MTase, 49 kDa) with a 20-mer oligonucleotide duplex containing the palindrome recognition site GGATCC was studied by methods of steady-state and presteady-state kinetics of the methyl group transfer, gel retardation, and crosslinking of the enzyme subunits with glutaric aldehyde. In steady-state conditions, BamHI MTase displays a simple kinetic behavior toward a 20-mer oligonucleotide substrate. A linear dependence was observed for the reaction rate on the enzyme concentration and a Michaelis dependence of the reaction rate on the concentration of both substrates: S-adenosyl-L-methionine (SAM), the methyl group donor, and DNA, the methyl group acceptor. In independent experiments, the concentration of the 20-mer duplex or SAM was changed, the enzyme concentration being substantially lower then the concentrations of substrates. The kcat values determined in these conditions are in good agreement with one another and approximately equal to 0.05 s-1. The Km values for the duplex and SAM are 0.35 and 1.6 microM, respectively. An analysis of single turnover kinetics (at limiting concentration of the 20-mer oligonucleotide duplex) revealed the following characteristics of the BamHI MTase-dependent methylation of DNA. The value of rate constant of the DNA methylation step at the enzyme saturating concentration is on average 0.085 s-1, which is only 1.6 times higher than the value determined in steady-state conditions. Only one of two target cytidine residues was methylated in the course of the enzyme single turnover, which coincides with the earlier data on EcoRI MTase. Regardless of the order of the enzyme preincubation with SAM and DNA, both curves for the single turnover methylation are comparable. These results are consistent with the model of the random order of the productive ternary enzyme-substrate complex formation. In contrast to the relatively simple kinetic behavior of BamHI MTase in the steady-state reaction are the data on the enzyme binding of DNA. In gel retardation experiments, there was no stoichiometrically simple complexes with the oligonucleotide duplex even at low enzyme concentrations. The molecular mass of the complexes was so high that they did not enter 12% PAG. In experiments on crosslinking of the BamHI MTase subunits, it was shown that the enzyme in a free state exists as a dimer. Introduction of substoichiometric amounts of DNA into the reaction mixture results in pronounced multimerization of the enzyme. However, addition of SAM in saturating concentration at an excess of the oligonucleotide duplex over BamHI MTase converts most of the enzyme into a monomeric state.     

In vitro DNA cytosine methylation of cis-regulatory elements modulates c-Ha-ras promoter activity in vivo.

The effect of DNA cytosine methylation on promoter activity was assessed using a transient expression system employing pHrasCAT. This 551 bp Ha-ras-1 gene promoter region is enriched with 84 CpG dinucleotides, six functional GC boxes, and is prototypic of many genes possessing CpG islands in their promoter regions. Bacterial modification enzymes HhaI methyl transferase (MTase) and HpaII MTase, alone or in combination with a human placental DNA methyltransferase (HP MTase) that methylates CpG sites in a generalized manner, including asymmetric elements such as GC box CpG's, were used to methylate at different types of sites in the promoter. Methylation of HhaI and HpaII sites reduced CAT expression by approximately 70%-80%, whereas methylation at generalized CpG sites with HP MTase inactivated the promoter by greater than 95%. The inhibition of H-ras promoter activity was not attributable to methylation-induced differences in DNA uptake or stability in the cell, topological form of the plasmid, or methylation effects in non-promoter regions.     

Molecular mechanisms of adaptive response to alkylating agents in Escherichia coli and some remarks on O(6)-methylguanine DNA-methyltransferase in other organisms

Alkylating agents are environmental genotoxic agents with mutagenic and carcinogenic potential, however, their properties are also exploited in the treatment of malignant diseases. O(6)-Methylguanine is an important adduct formed by methylating agents that, if not repaired, can lead to mutations and death. Its repair is carried out by O(6)-methylguanine DNA-methyltransferase (MTase) in an unique reaction in which methyl groups are transferred to the cysteine acceptor site of the protein itself. Exposure of Escherichia coli cells to sublethal concentrations of methylating agents triggers the expression of a set of genes, which allows the cells to tolerate DNA lesions, and this kind of inducible repair is called the adaptive response. The MTase of E. coli, encoded by the ada gene was the first MTase to be discovered and one of best characterised. Its repair and regulatory mechanisms are understood in considerable detail and this bacterial protein played a key role in identification of its counterparts in other living organisms. This review summarises the nature of alkylation damage in DNA and our current knowledge about the adaptive response in E. coli. I also include a brief mention of MTases from other organisms with the emphasis on the human MTase, which could play a crucial role in both cancer prevention and cancer treatment.